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Tumor-infiltrating lymphocytes isolation for flow cytometry analysis

XX Xiangling Xiao
JS Jie Shi
CH Chuan He
XB Xia Bu
YS Yishuang Sun
MG Minling Gao
BX Bolin Xiang
WX Wenjun Xiong
PD Panpan Dai
QM Qi Mao
XX Xixin Xing
YY Yingmeng Yao
HY Haisheng Yu
GX Gaoshan Xu
SL Siqi Li
YR Yan Ren
BC Baoxiang Chen
CJ Congqing Jiang
GM Geng Meng
YL Yu-Ru Lee
WW Wenyi Wei
GF Gordon J. Freeman
CX Conghua Xie
JZ Jinfang Zhang
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Freshly resected tumors from mice were first mechanically minced into about 1–3 mm pieces and chemically digested in dissociation buffer (1 mg/mL collagenase IV (BS156, Biosharp), 0.5 mg/mL collagenase I (BS153, Biosharp), 0.002 mg/mL hyaluronidase (BS171, Biosharp) in HBSS) with rotation at 37 °C for 30 min. After passing through a 70 μM strainer, red blood cells in the single-cell suspensions were lysed with ACK lysis buffer (A1049201, Thermo Fisher). To analyze T cell effector function, cells were incubated with appropriate antibodies for 30 min at 4 °C in dark. Flow cytometry was performed on FACSCelesta (BD Biosciences), and data were analyzed using Flowjo 10.6.2 software (TreeStar).

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